RESEARCH ARTICLE

Cdc42 regulates epithelial cell polarity and cytoskeletal functionduring kidney tubule developmentBertha C. Elias1, Amrita Das2, Diptiben V. Parekh1, Glenda Mernaugh1, Rebecca Adams1, Zhufeng Yang2,Cord Brakebusch3, Ambra Pozzi1,4,5, Denise K. Marciano2, Thomas J. Carroll2,6 and Roy Zent1,4,7,*

ABSTRACTThe Rho GTPase Cdc42 regulates key signaling pathways requiredfor multiple cell functions, including maintenance of shape, polarity,proliferation, migration, differentiation and morphogenesis. Althoughprevious studies have shown that Cdc42 is required for properepithelial development and maintenance, its exact molecular functionin kidney development is not well understood. In this study, we definethe specific role of Cdc42 during murine kidney epithelialtubulogenesis by deleting it selectively at the initiation of uretericbud or metanephric mesenchyme development. Deletion in eitherlineage results in abnormal tubulogenesis, with profound defects inpolarity, lumen formation and the actin cytoskeleton. Ultimately, thesedefects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processesby regulating the polarity Par complex (Par3–Par6–aPKC–Cdc42)and the cytoskeletal proteins N-Wasp and ezrin. Thus, we concludethat the principal role of Cdc42 in ureteric bud and metanephricmesenchyme development is to regulate epithelial cell polarity andthe actin cytoskeleton.

KEY WORDS: Ureteric bud, Metanephric mesenchyme, GTPase,Signaling, Epithelial cell

INTRODUCTIONMammalian kidney development begins when the ureteric budinvades the surrounding metanephric mesenchyme (Carroll andDas, 2013; Costantini and Kopan, 2010; Dressler, 2006). Theureteric bud is induced to undergo iterative rounds of branchingmorphogenesis and gives rise to the collecting system, whereas themetanephric mesenchyme forms the nephrons that consist of theglomeruli and renal tubules. Reciprocal signaling betweenthe ureteric bud and the metanephric mesenchyme is requiredfor ureteric bud branching morphogenesis and metanephricmesenchyme development.Cdc42, a member of the Rho GTPases, plays a crucial role in

regulating multiple cell functions including cell shape, polarity,proliferation, invasion, migration and differentiation (Melendezet al., 2011). Cdc42 mediates these functions by controlling

cytoskeletal dynamics due to its ability to interact with the Wiskott–Aldrich syndrome protein (Wasp) and p21-activated kinases(PAKs). Wasp regulates actin polymerization and filopodiaformation through direct interactions with both profilin and actin,and PAKs alter the activity of the crucial actin-binding proteincofilin (Melendez et al., 2011). In addition, Cdc42 regulatesepithelial cell polarity by forming complexes with the PAR proteinfamily, atypical protein kinase C (aPKC) and cadherins (Goldsteinand Macara, 2007).

Both in vitro and in vivo evidence suggest a crucial role for Cdc42in kidney development. Cdc42 is required by Madin–Darby caninekidney (MDCK) cells to form tubules in three-dimensional (3D)culture by regulating the formation of the apical plasma membranedomain and tight junctions as well as primary ciliogenesis (Choiet al., 2013; Kim et al., 2007; Martin-Belmonte et al., 2007;Rodriguez-Fraticelli et al., 2010; Rogers et al., 2003; Zhang et al.,2001; Zuo et al., 2011). Two studies have shown that Cdc42 isessential for normal kidney development and function. In the first,Cdc42 deletion from renal tubular epithelia with the Ksp-cre (whichcauses cre-mediated recombination in developing nephrons and therenal collecting system) was shown to result in lethal cystic kidneydisease that was attributed to abnormalities in ciliogenesis (Choiet al., 2013). In a second study, Cdc42 was deleted in the nephronprogenitors using Six2-cre. These mice formed hypoplastic kidneyswith a reduced nephrogenic zone and small papillae (Reginensiet al., 2013). This phenotype was attributed to defects in localizationof the transcriptional activator Yap. Although both studiesdemonstrate roles for Cdc42 in kidney development, theirproposed mechanisms of action for Cdc42 are different and out ofkeeping with its principal function as a master regulator of the actincytoskeleton and a mediator of epithelial cell polarity.

There is little known about how apical-basal polarity isestablished during renal tubulogenesis. Afadin, an F-actin-binding and nectin adaptor protein, has been shown to beessential for the timely initiation of apical-basal polarity duringepithelialization of developing nephrons (Yang et al., 2013). Inaddition, this adaptor protein is essential for the formation of acontinuous apical surface and lumen formation (Yang et al., 2013).At the molecular level, absence of afadin in developing nephronsresults in a delayed lumen formation and a reduced ability tocorrectly localize members of the Par complex (Par3–Par6–aPKC–Cdc42; in mammals, Par3 is also known as PARD3 and Par6 asPARD6, with each having two isoforms) to the forming epithelia,suggesting that defects in Par complex function and Cdc42activation might be the underlying defect. Afadin is necessary forCdc42 activation in vitro (Kawakatsu et al., 2002; Kurita et al.,2013; Mandai et al., 2013; Nakanishi and Takai, 2004), suggestingthat the principal mechanisms whereby Cdc42 regulatestubulogenesis in the kidney might be by altering the actincytoskeleton and apical-basal polarity of the epithelial cells.Received 10 October 2014; Accepted 14 October 2015

1Division of Nephrology and Hypertension, Department of Medicine, VanderbiltUniversity School of Medicine, Nashville, TN 37232, USA. 2Department ofMedicine, University of Texas Southwestern Medical Center, Dallas, TX 75390,USA. 3Biotech Research Center, University of Copenhagen, Ole Maaløes Vej 5,Copenhagen DK-2200, Denmark. 4Department of Cancer Biology, VanderbiltUniversity School of Medicine, Nashville, TN 37232, USA. 5Veterans AffairsHospital, Nashville, TN 37232, USA. 6Department of Molecular Biology, Universityof Texas Southwestern Medical Center, Dallas, TX 75390, USA. 7Department of Celland Developmental Biology, Vanderbilt University School of Medicine, Nashville,TN 37232, USA.

*Author for correspondence (roy.zent@vanderbilt.edu)

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Given the discrepant findings of Cdc42 function in renalepithelial tubulogenesis compared to its demonstrated function inother epithelia, and the recent findings suggesting that afadinregulates Cdc42 and renal epithelial cell polarity, we investigatedthe role of Cdc42 in the developing kidney, focusing on its role inestablishing apical-basal polarity and in cytoskeletal architecture.We used Cdc42flox/flox mice crossed with two different cre deletorstrains; Six2-cre and Hox-B7-cre. Six2-cre deletes Cdc42 in the capmesenchyme of the metanephric mesenchyme and was used in oneof the prior studies (Reginensi et al., 2013). Hoxb7-cre deletesCdc42 in the ureteric bud, which gives rise to the collecting systemstarting at embryonic day (E)10.5. We show that Cdc42 is requiredfor both ureteric bud and metanephric mesenchyme development asthe Hoxb7-cre:Cdc42flox/flox mice develop a severe branchingmorphogenesis defect, and Six2-cre:Cdc42flox/flox mice are unableto form nephrons. Both mice have serious abnormalities in epithelialcell polarization at all developmental stages examined. In particular,the Six2-cre:Cdc42flox/flox kidney phenotype is highly reminiscentof the afadin mutants with disruption of Par complex formation anddiscontinuous lumens. Using Cdc42-null collecting duct cells, weshowed that absence of Cdc42 caused profound adhesion,migration, and polarization defects, as well as marked reductionin N-Wasp (also known as WASL) and ezrin activation. Thus, weconclude that a primary function of Cdc42 in the formation of

kidney tubules is to promote epithelial cell polarization through Parcomplex formation and to regulate cytoskeletal architecture througha Cdc42–N-Wasp–ezrin signaling pathway.

RESULTSCdc42 is required for ureteric bud developmentWe crossed Hoxb7-cre mice, which express cre recombinase inthe Wolffian duct and ureteric bud from E10.5 onwards, withCdc42flox/flox mice to define the role of Cdc42 in the developingureteric bud. The mice were born in the predicted Mendelian ratio;however, all the Hoxb7-cre:Cdc42flox/flox mice died by four weeksof age. Cdc42 deletion was confirmed in mutant mice byperforming immunohistochemistry on E15 embryos (Fig. 1A,B)and immunoblotting on isolated papillae of newborn Hoxb7-cre:Cdc42flox/flox with an antibody directed at Cdc42 (Fig. 1C). Thekidneys of the Hoxb7-cre:Cdc42flox/flox mice were much smallerthan their wild-type controls and had histological features of end-stage renal disease characterized by severe destruction of the cortexand medulla (Fig. 1D–I). When Hoxb7-cre:Cdc42flox/flox mice werekilled prior to end-stage renal failure; there was relative preservationof the cortex compared to the severe tubular abnormalities in themedulla. The markedly decreased number of tubules foundwithin the renal papilla of the dysmorphic dysplastic Hoxb7-cre:Cdc42flox/flox kidneys were consistent with a major branching

Fig. 1. Hoxb7-cre:Cdc42flox/flox mice developend-stage renal failure due to defectivebranching morphogenesis and intraluminalobstruction in the collecting ducts.(A–C) Sections of E15.5 mouse kidneys fromCdc42flox/flox and Hoxb7-cre:Cdc42flox/flox micewere stained with antibodies directed againstCdc42. NoCdc42was visualized in the developingureteric bud (arrow) of Hoxb7-cre:Cdc42flox/flox

mice (A,B). Deletion of Cdc42 in the Hoxb7-cre:Cdc42flox/flox mice was confirmed byimmunoblotting the kidney papilla of newbornmicewith an anti-mouse Cdc42 antibody (C).(D–I) Microscopy of hematoxylin and eosin(H&E)-stained kidney slides showing destructionof the medulla and corticomedullary junction inHoxb7-cre:Cdc42flox/flox but not in the Cdc42flox/flox

kidneys. Total destruction of the medulla due tohydronephrosis in Hoxb7-cre:Cdc42flox/flox mice ispresent just prior to death at 4 weeks of age (F); at2 weeks of age the kidneys are severelyhypoplastic and dysplastic with dilated tubulesfound within the cortex and medulla and evidenceof focal tubular cysts (G). High power images(H) show the medulla where the lumens of thecollecting ducts are filled with cells (arrows) andthe tubules are dilated. Dilated tubules andinterstitial fibrosis (*) are seen in the cortex (I). Themagnification at which the images were viewed isdisplayed.

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morphogenesis defect (Fig. 1G). Many lumens of the collectingducts were not apparent (marked by arrows in Fig. 1H) and therewere also areas of marked interstitial fibrosis between the dilatedcortical collecting ducts (marked by * in Fig. 1I).We next defined when the branching defect in the mice began by

performing histological studies of kidneys at various embryonicstages. As early as E12.5, there were fewer ureteric bud structures(marked by arrows) and less metanephric mesenchyme induction inmutants compared to wild type (Fig. 2A,B). This became moreevident over time. The mutant ureteric bud was very poorly formedat E15.5 (Fig. 2C,D) and E18.5 (Fig. 2E,F) with fewer nephrons atboth these time points. The branching defect was confirmed bycalbindin staining at E18.5 (Fig. 2G,H). To define why the kidneysof the Hoxb7-cre:Cdc42flox/flox mice were hypoplastic, we

quantified proliferation and apoptosis in the kidney. Interestinglythere was no statistical difference in proliferation of the cytokeratin-positive ureteric bud cells in the Hoxb7-cre:Cdc42flox/flox kidneys asdefined by staining for phosphorylated histone H3 (pHH3) at E13.5,E15.5 or E18.5 (Fig. 2I–K). In contrast, when we assessedproliferation of Six2 positive cells at E18.5, there was a 60%decrease in the Hoxb7-cre:Cdc42flox/flox kidneys (Fig. 2I,J,L) andthis difference was also seen in newborn mice (data not shown).There was no difference in apoptosis rates between the twogenotypes at any time points (data not shown). Thus, deletion ofCdc42 in the developing ureteric bud caused a severe ureteric budbranching defect and secondarily impaired cell proliferation withinthe metanephric mesenchyme, which presumably contributed to thereduced kidney size.

Fig. 2. Hoxb7-cre:Cdc42flox/flox mice have a severe branchingmorphogenesis defect and impairedmetanephric mesenchyme induction. (A–H) Kidneyswere isolated from embryos of the Cdc42flox/flox and the Hoxb7-cre:Cdc42flox/flox mice at E12.5 (A,B), E15.5, (C,D) and E18.5 (E–J). A marked ureteric budbranching morphogenesis defect (arrows) and decreased metanephric mesenchyme induction was noted at each of these time points. Calbindin stainingdemonstrated a decrease in ureteric bud structures at E18.5 in the Hoxb7-cre:Cdc42flox/flox mice when compared to the Cdc42flox/flox mice (G,H). pHH3 stainingwas performed on cytokeratin-positive cells (marking ureteric bud structures, red) and Six2-positive cells (marking metanephric mesenchyme structures, blue)(I,J). The percentage of proliferation at different time points in the ureteric bud (K) and metanephric mesenchyme (L) was quantified (mean±s.d., at least threemice were examined per group). *P<0.05 for differences between Cdc42flox/flox and the Hoxb7-cre:Cdc42flox/flox in the Six2-positive cells (Student’s t-test). Themagnification at which the images were viewed is displayed.

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Cdc42 is required for metanephric mesenchymedevelopmentCdc42 was deleted from the metanephric mesenchyme by crossingthe Cdc42flox/flox with the Six2-cre mouse. Six2 is expressed in thecap mesenchyme and this Cre line deletes floxed genes from allcomponents of the developing nephron extending from the

glomerular epithelium to the connecting segment (Kobayashiet al., 2008). We verified that Cdc42 was deleted in thedeveloping metanephric mesenchyme by performingimmunoblotting and immunohistochemistry on post-natal day (P)1 Cdc42flox/flox and Six2-cre:Cdc42flox/flox mice (Fig. 3A–C).Staining revealed clearly decreased Cdc42 expression in the

Fig. 3. Six2-cre:Cdc42flox/flox mice have adefect in metanephric mesenchymedevelopment. (A) Deletion of Cdc42 in theSix2-cre:Cdc42flox/flox mice was confirmed byimmunoblotting extracts from the kidney cortexof newborn mice with an anti-mouse-Cdc42antibody. (B,C) No staining with an anti-Cdc42antibody was seen in the metanephricmesenchyme of newborn Six2-cre:Cdc42flox/flox

mice. (D,E) No ureteric bud branching defectwas seen in the E12.5 Six2-cre:Cdc42flox/flox

mice. (F–I) Diminished metanephricmesenchyme development was seen in E18.5Six2-cre:Cdc42flox/flox mice (F,G), which waseven more severe at P1 (H,I) with almost nonephrons formed (arrow) in the metanephricmesenchyme. (J,K) The diminishedmetanephric mesenchyme development wasverified in E18.5 Six2-cre:Cdc42flox/flox kidneysas decreased lotus tetragonolobus lectin (LTL),a marker of proximal tubules, staining.(L–N) pHH3 staining was performed onSix2-positive cells of E15.5 kidneys fromCdc42flox/flox (L,M)and theSix2-cre:Cdc42flox/flox

mice and quantified (mean±s.d., at least threemicewere examined per group) (N). *P<0.01 fordifferences between Cdc42flox/flox and theSix2-cre:Cdc42flox/flox (Student’s t-test). Themagnification at which the images were viewedis displayed.

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cortical kidney tissue of Six2-cre:Cdc42flox/flox mice. Because allthe Six2-cre:Cdc42flox/flox mice died within 24 h of birth, wedefined when the kidney abnormalities first became evident.Although the kidneys appeared normal at E12.5 (Fig. 3D,E),decreased metanephric mesenchyme development was evident byE15 (data not shown). This defect was clearly obvious at E18.5(Fig. 3F,G) and P1, where there were markedly decreased numbersof renal vesicles, and comma- and S-shaped bodies (Fig. 3H,I). Thefew glomeruli that were seen in the mutant mice were hypoplasticand dysplastic. Mutant pups died within a few hours of birth,presumably due to lack of filtration through the hypoplastic anddysplastic nephrons. Lotus Tetragonolobus Lectin (LTL)-positiveproximal tubules were greatly reduced in mutants althoughthey were evident and abundant in wild-type controls at E18.5(Fig. 3J,K). There was 40% less proliferation of Six2-positive cellsas defined by pHH3 staining cells in the metanephric mesenchymeof Six2-cre:Cdc42flox/flox kidneys at E15.5 when compared toCdc42flox/flox kidneys (Fig. 3L–N) and there were no differences inapoptosis between the two genotypes (data not shown). Thus,removing Cdc42 in the metanephric mesenchyme resulted in severehypoplasia and dysplasia of the developing renal vesicles and aprofound proliferation defect.

Cdc42 is required for normal polarization and differentiationof the ureteric budOne of the features of the kidneys of the Hoxb7-cre:Cdc42flox/flox

mice was obstruction of the tubular lumens (Fig. 1), whichsuggested polarity defects in the tubular epithelial cells. As amajor function of Cdc42 is to regulate epithelial polarity, we definedwhether there were abnormalities in expression of the highlyconserved polarity proteins that form the Par complex found in theapical domain of polarized epithelial cells. To do this, we performedimmunostaining of two key components of the complex, Par3 andatypical protein kinase C (aPKC) in Hoxb7-cre:Cdc42flox/flox mice.There was decreased apical staining of aPKC and Par3 in all cellsderived from the ureteric bud (E-cadherin, cytokeratin-doublepositive epithelia) in Hoxb7-cre:Cdc42flox/flox mice at E15.5(Fig. 4A–D) and E18.5 (Fig. S1A–D), suggesting that there is apolarity defect in the developing ureteric bud that persists over time.Consistent with these apical polarity abnormalities, there wasmarkedly decreased expression of the tight junction protein ZO-1(also known as TJP1) in the Hoxb7-cre:Cdc42flox/flox mice(Fig. S1E,F). The cells within the developing ureteric bud of theHoxb7-cre:Cdc42flox/flox mice also had severe defects of terminaldifferentiation as they did not express the apical water

Fig. 4. Localization of apical proteins in the collecting ducts of wild-type and Cdc42 mutant kidneys at E15.5. Immunostaining of E15.5 Cdc42flox/flox

(A,A′,A″,C,C′,C″) and Hoxb7-cre:Cdc42flox/flox (B,B′,B″,D,D′,D″) kidneys stained with antibodies to aPKC (green in A,A′,B,B′), Par3 (green in C,C′,D,D′),E-cadherin (red in A″,B″,C″ and D″) and cytokeratin (CK) (white in in A‴,B‴,C‴ and D‴). A,B,C and D are merged panels, with the individual channels beingshown in the right-hand panels. All sections have been stained with DAPI (blue in A,B,C,D) to show the nuclei. Scale bars: 20 μm.

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channel aquaporin-2 (Aqp2), whereas the Cdc42flox/flox mice did(Fig. S1E,F). To verify these terminal differentiation defects of thecollecting duct were generalized, we investigated whether there wasan altered expression level of the vacuolar-type H ATPase, which isexpressed exclusively in the intercalated cells of the collecting duct.This protein was not detectable in the Hoxb7-cre:Cdc42flox/flox mice(Fig. S2A,B). Finally, we also measured the number of cilia presentin the Hoxb7-cre:Cdc42flox/flox mice as Cdc42 has been shown tobe required for cilia production (Zuo et al., 2011). There were50% fewer cilia in Hoxb7-cre:Cdc42flox/flox mice compared toCdc42flox/flox mice (Fig. S2C–E). Taken together, these dataindicate that the polarized epithelial cells in the ureteric bud ofthe Hoxb7-cre:Cdc42flox/flox mice have a profound polarity andterminal differentiation defect, which is associated with decreasedprimary cilia. These cellular abnormalities likely cause intraluminaltubular obstruction that subsequently results in dilated tubules andobstruction of the kidney.

Defects in metanephric mesenchyme epithelial cell polarityresults in abnormal renal vesicle formationDuring nephrogenesis, mesenchymal nephron progenitors undergocompaction to form a pretubular aggregate of cells that subsequentlyform a polarized sphere of epithelia called the renal vesicle, whichcontains a central lumen. In pretubular aggregates, neural cell

adhesion molecule (NCAM, also known as NCAM1) marks theplasma membrane, but becomes restricted to basolateral membranesas a renal vesicle lumen is formed. Pretubular aggregates have littleaPKC, and Par-3 is found in dispersed short segments of plasmamembrane, called ‘pre-apical domains’ (Yang et al., 2013). As therenal vesicle lumen forms, both aPKC and Par3 become abundant atthe apical surface, with Par-3 becoming redistributed to apical andlateral junctions shortly thereafter (Yang et al., 2013). We found thatwhereas Six2-cre:Cdc42flox/flox kidneys do form NCAM-positivepre-tubular aggregates, these structures do not progress to a structurewith a patent lumen by E15.5 (Fig. 5) and E18.5 (Fig. S3). aPKC isgreatly reduced or absent from many cells, whereas NCAM remainsrelatively uniform in its plasma membrane localization. Similar towhat is observed for aPKC, many structures at this stage lack Par-3or contain only small foci of Par-3. As demonstrated previously(Reginensi et al., 2013), deletion of Cdc42 in Six2-positive cellsresulted in a marginal reduction in total YAP (also known as YAP1)expression in the metanephric mesenchyme (Fig. S4A,B, arrow).The localization of the phosphorylated form of YAP, however,remained unchanged (Fig. S4C,D). Thus, Cdc42 is needed for thetimely initiation of lumen formation during renal vesicle formationand it is associated with reduced YAP expression.

We also examined the next stage of nephrogenesis, when therenal vesicle elongates into a primordial tubule called the S-shaped

Fig. 5. Cdc42 is essential for timely lumen formation indeveloping nephrons. Immunostaining of E15.5 kidneysfrom Cdc42flox/flox (A–A″; C–C″) and Six2-cre:Cdc42flox/flox

(B–B″,D–D″) with NCAM (red), aPKC or Par3 (green),E-cadherin (white) and DAPI (blue) as indicated. Controlsshow luminal clearing of nuclei with apical aPKC, basolateralNCAM and apical-lateral Par3. In contrast, mutants have noevidence of a central clearing of nuclei, display relativelyuniform NCAM with little or no aPKC. Mutants also havereduced Par3, with some mutant pretubular aggregate orrenal vesicle structures showing none or only small foci ofPar3. Results are representative of sections from at least twomice. Similar results were obtained at E18.5 (Fig. S2). Arrowshighlight the renal vesicle. RV, renal vesicle; PA, pretubularaggregate; UB, ureteric bud. Ureteric buds and renal vesiclesor pretubular aggregates are outlined by a white dotted line.Scale bars: 10 μm.

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body. In the Cdc42flox/flox kidneys, the elongated S-shaped tubulefuses with the tubule of the ureteric bud and forms a continuouslumen throughout the S-shaped body and ureteric bud at this stage.By contrast, in Six2-cre:Cdc42flox/flox kidneys there is evidence oflumen formation, but the lumens are short and discontinuous despitethe apparent fusion of the S-shaped body to the ureteric bud (Fig. 6).The lumens of mutants remain discontinuous even in the later stagesof the formation of the S-shaped body, suggesting it is not simply adelay in theirability to inter-connect. From these studies,weconcludethat Cdc42 is required not only for timely lumen initiation, butalso for continuous lumen formation during kidney morphogenesis.

Collecting duct cells lacking Cdc42 are unable to formtubules and have polarity defectsOur in vivo findings suggested that deleting Cdc42 resulted insevere abnormalities in tubulogenesis in both the metanephricmesenchyme and the ureteric bud due at least in part to polaritydefects. To determine the mechanism of this deficiency, we isolatedcollecting duct cells from Cdc42flox/flox mice and deleted Cdc42in vitro using adenoviral-mediated delivery of a plasmid encodingCre recombinase. We completed serial dilutions on cell populationsand identified cells that did not express Cdc42 by immunoblotting(Fig. 7A). We initially performed experiments on at least threedifferent populations to show that the results obtained were not dueto clonal selection (data not shown). When Cdc42flox/flox collecting

duct cells were placed in 3D collagen-I or matrigel gels, they wereunable to form tubules and few cells were observed under theseconditions (Fig. 7B,C). Mechanistically, collecting duct cells needto be able to adhere, spread and migrate through extracellular matrixto form tubules. We therefore defined which of these processes weredefective in the Cdc42−/− collecting duct cells. We identified asevere adhesion defect in the Cdc42−/− collecting duct cells oncollagen I, laminin-111 (data not shown) and -511 (data not shown)and fibronectin (Fig. 7F), although the cells had normal expressionof integrin β1, α1, α2, α3, α5 and α6 subunits (data not shown).Thus, deleting Cdc42 altered cell adhesion of collecting duct cellson multiple extracellular matrix (ECM) components, suggesting thisadhesion defect was not integrin specific. Consistent with theadhesion defects, these cells had a profound haptotactic migrationdefect on collagen 1, laminin-111 (data not shown) and -511 (datanot shown) and fibronectin (Fig. 7G). They also had a majorspreading defect on collagen 1 (Fig. 7D,E), laminin-111 and -511and fibronectin (data not shown). Owing to the small number ofcells seen in the Cdc42−/− collecting duct cell tubulogenesis assay,we performed proliferation assays of these cells on multiple ECMsincluding collagen 1 and fibronectin under low-serum conditions. Inagreement with the results seen in vivo and in the in vitro 3D assays,there was a marked decrease in Cdc42−/− collecting duct cellproliferation that was independent of the ECM substrate (Fig. 7H).Thus, Cdc42 deletion from collecting duct cells results in a severe

Fig. 6. Cdc42 is required forcontinuous lumen formation innephron tubules. Shown are 2Dimages and 3D reconstructions fromconfocal z-stacks of E15.5 kidneysimmunostained with anti-aPKCantibody (green), F-actin (phalloidin,blue) and anti-NCAM (red) antibodyas indicated. aPKC marks the apicalsurface, thereby demarcating thelumen. NCAM delineates basolateralcell membranes of developingnephron tubules. The S-shaped bodystage of nephron tubulogenesis isshown in Cdc42flox/flox (A–A″) andSix2-cre:Cdc42flox/flox (B–C″) mice.Images from A and B show S-shapedbodies (SB) at a similar stage,highlighting the discontinuousS-shaped body lumen in mutants(arrows). Images from C show a lateS-shaped nephron, illustrating thatthe discontinuous lumens persist inmutants. Results are representativeof sections from at least two mice.Abbreviations: SB, S-shaped body;UB, ureteric bud. The S-shaped bodyis outlined by a white dotted line.Scale bars: 10 μm.

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collecting duct cell tubulogenesis defect due to decreased adhesion,spreading, migration and proliferation.Mechanistically, Cdc42 regulates multiple cellular processes,

including polarization, actin cytoskeletal organization and cellsignaling. Based on our in vivo and in vitro data, we initiallyinvestigated whether the Cdc42−/− collecting duct cells had a

polarization defect by growing them on Transwell inserts andperforming immunostaining for the adherens junction proteinE-cadherin, and the tight junction protein ZO-1. Much less of boththese proteins localized to the cell–cell junctions of the Cdc42−/−

collecting duct cells when compared to Cdc42fl/fl collecting duct cells(Fig. 7I–L). We next employed biochemical techniques to determine

Fig. 7. Deleting Cdc42 from collecting duct cells results in abnormal tubulogenesis, adhesion, migration, proliferation and polarity defects.(A) Immunoblotting was performed on Cdc42flox/flox and Cdc42−/− cell extracts to verify Cdc42 deletion. (B,C) Cdc42flox/flox and Cdc42−/− collecting duct cells wereplaced into 3D collagen and Matrigel gels and allowed to undergo tubulogenesis over 7 days in the presence of 5% FBS. They were stained with Rhodamine–phalloidin and visualized by confocal microscopy. (D,E) Cdc42flox/flox and Cdc42−/− collecting duct cells were plated on collagen I and allowed to spread for 1 h,after which theywere stained with Rhodamine–phalloidin. (F) Cdc42flox/flox and Cdc42−/− collecting duct cells populations were allowed to adhere to collagen I andfibronectin (10 μg/ml) and cell adhesion was evaluated 1 h after plating. (G) Cdc42flox/flox and Cdc42−/− collecting duct cells were plated on Transwells coated withcollagen I (10 μg/ml) or fibronectin (10 μg/ml) andmigration was evaluated after 4 h. This was quantified and expressed as the number of cells per high-power field(cells/hpf ). (H) Cdc42flox/flox and Cdc42−/− collecting duct cells were plated on collagen I. After 24 h, cells were treated with 3H-thymidine and incubated for afurther 24 h. 3H-thymidine incorporation was then determined and expressed as counts per minute (cpm) as described in the Materials and Methods. In F–H,values are mean±s.d. from three experiments performed in triplicate. *P<0.01 for the difference between Cdc42flox/flox and Cdc42−/− collecting duct cells(Student’s t-test). (I–L) Cdc42flox/flox and Cdc42−/− collecting duct cells were grown to confluence on Transwell inserts and immunostained for ZO-1 andE-cadherin. (M,N) Equal amounts of whole cell lysate from Cdc42flox/flox and Cdc42−/− collecting duct cells were electrophoresed and immunoblotted forE-cadherin, ZO-1, Par3, aPKC and β-actin. The intensities of a single blot were measured and shown on a graph (N), which is representative of at least threesimilar experiments. (O–Q) Equal amounts of Triton-soluble (TS) and -insoluble (TI) fractions from Cdc42flox/flox and Cdc42−/− collecting duct cells were analyzedby western blotting for E-cadherin, ZO-1, Par3, aPKC and β-actin. The intensities of a single blot were measured and shown graphically (P,Q). This isrepresentative of at least three similar experiments.

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whether there was decreased expression or mislocalization ofE-cadherin, ZO-1 and the apical proteins Par3 and aPKC in theCdc42−/− collecting duct cells. There was marked decrease ofE-cadherin, ZO-1 and Par3 and a moderate decrease of aPKC in theCdc42−/− collecting duct cells (Fig. 7M,N). When we analyzed therelative amounts of these proteins inTriton-soluble and -insoluble cellfractions, there was less E-cadherin, ZO-1, aPKC and Par3 in theTriton-insoluble (membranous) fraction of Cdc42−/− collecting ductcell lysates relative to Cdc42fl/fl (control) cell lysates (Fig. 7O–Q).Taken together, these data demonstrate a polarity defect in Cdc42−/−

collecting duct cells, which is consistent with our in vivo findings.

Cdc42−/− regulates the actin cytoskeleton in the collectingsystem of the kidneyThe severe adhesion, migration and spreading defect manifested bythe Cdc42−/− collecting duct cells suggested they have abnormalintegrin-dependent signaling. When we investigated this possibility,no differences between Cdc42f/f and Cdc42−/− collecting duct cellswere found when we assessed activation of well recognizedsignaling proteins such as those of the Akt or ERK family that actdownstream of integrins using re-plating assays on collagen 1 orlaminin-111 and -511 (data not shown). Cdc42 has also beenreported to be a key regulator of cytoskeletal proteins, such asN-Wasp, which activates the Arp2/3 complex, and ezrin, whichbinds N-Wasp (Albiges-Rizo et al., 2009). When we definedhow Cdc42 regulated the activation of these proteins in responseto glial-derived neurotropic factor (GDNF), a principal growthfactor responsible for ureteric bud branching morphogenesis,phosphorylation of both these proteins was severely decreased inCdc42−/− compared to Cdc42fl/fl collecting duct cells, despite thesame amount of activation of the Ret receptor (Fig. 8A). These datasuggest that the severe adhesion, spreading and migration defectsobserved in the Cdc42−/− collecting duct cells, are due at least inpart to the requirement of Cdc42 for N-Wasp and ezrin activation,and that this signaling defect is not due to decreased Ret activation.We next examined whether an actin cytoskeleton defect was

present in vivo by staining the developing ureteric bud at E15.5with Rhodamine–phalloidin. Interestingly there was no obviousstructural abnormality in the subapical distribution of actin in theHoxb7-cre:Cdc42flox/flox mice (Fig. 8B,C). The subapical actin alsoappeared normal in distribution in the Six2-cre:Cdc42flox/flox miceexcept in the areas in which there was no discernible lumen (Fig. 6).As a lack of obvious structural defects does not necessarily indicatenormal actin cytoskeleton function, we examined the distribution ofezrin. In Cdc42flox/flox mice ezrin staining was clearly expressed inthe apex of the lumen of the ureteric bud; however, this staining wasdecreased and diffuse at the apical surface of collecting ducts in theHoxb7-cre:Cdc42flox/flox mice (Fig. 8D,E), which is consistent withour in vitro data. Taken together, these data demonstrate that Cdc42is required for apical actin cytoskeletal function in developingcollecting ducts, and for N-Wasp and ezrin signaling in collectingduct cells in vitro.

DISCUSSIONCdc42 has diverse cellular functions and plays a role indevelopment of many organs. We show that the absence of Cdc42in the ureteric bud causes severe branching abnormalities aswell as profound polarity and cytoskeletal defects that causemalformed and obstructed tubular lumens. Similarly, deletingCdc42 from the developing metanephric mesenchyme producessevere abnormalities in epithelial polarity and lumen formationresulting in failed renal vesicle and S-shaped body establishment.

We confirmed the requirement for Cdc42 for tubule formationin vitro by utilizing Cdc42-null collecting duct cells, which alsoshowed severe adhesion, migration, proliferation, polarization andcytoskeletal defects. Thus, we conclude that the principal role ofCdc42 in ureteric bud and metanephric mesenchyme developmentis to regulate epithelial cell polarity and actin cytoskeleton function.

It has previously been shown that deletion of Cdc42 in the distaltubule of the kidney using Ksp-cre induced fatal kidney failure;however, the few surviving mice developed cystogenesis associatedwith decreased ciliogenesis and interstitial fibrosis (Choi et al.,2013). Most of the characterization of these mice was performedwithin the cysts where increased cell proliferation and apoptosisassociated with increased MAPK pathway activation was noted. Noepithelial cell polarization defects were observed, as measured byE-cadherin or gp135 (also known as CNTN1) localization. Thediscrepancies between this and our studies could be attributed to thefact that Hoxb7-cre deletes Cdc42 at the initiation of ureteric budbranching morphogenesis in the newly forming bud tips at E10.5(Yu et al., 2002). In contrast, Ksp-cre deletes at E12.5 in the stalkregion of the ureteric bud, and from E15.5 onward, in more distalsegments of the nephron (Karner et al., 2009; Shao et al., 2002). It isnot clear whether the cysts observed in the Ksp-cre mutants are inthe collecting duct or the distal tubule. It is interesting to note thatsimilar to Choi et al. (2013), we did observe a decrease in cilianumber in mutant collecting ducts. This cellular phenotype was notas penetrant as the defects in polarity and differentiation, suggestingthat it might be a secondary manifestation of Cdc42 deletion.

Our morphological results of the Six2-cre:Cdc42flox/flox in thedeveloping metanephric mesenchyme are very similar to thosereported byReginensi and colleagues (2013), in that the kidneyswereseverely hypoplastic with almost no functional nephrons. Theyconcluded that the principal mechanism whereby Cdc42 regulatedmetanephric mesenchyme development was by altering thecytoskeleton through an N-WASP-dependent mechanism thataltered the nuclear localization of YAP. Furthermore, they did notcomment on amajor proliferation or polarity defect and they reportedno changes in Par3 or aPKC localization. Indeed, we did find thatdeletion of Cdc42 in Six2-positive cells resulted in a marginalreduction in total YAP expression in the metanephric mesenchyme(Fig. S4A,B, arrow), although the expression of the phosphorylatedform ofYAPappeared unchanged (Fig. S4C,D). Our study identifiedseveral other major cellular defects, including abnormalities inepithelial cell polarity and proliferation, tubule lumen formation, andactin cytoskeletal function.We postulate these are the primary causesof the defects in terminal differentiation of both the nephron and thecollecting duct epithelia. Our study is consistent with the findings inother branching organs, such as the pancreas (Kesavan et al., 2009)and lung (Wan et al., 2013), where epithelial deletion of Cdc42 hasbeen shown to be required for branching, epithelial cell proliferation,tubule formation, normal apical polarity and actin cytoskeletonformation as well as terminal differentiation and cell fatespecification. In addition, hepatocytes from Cdc42-null mice areunable to proliferate in response to hepatectomy (Yuan et al., 2009)and deleting Cdc42 from the colon results in a mild defect in apicaljunction orientation and impaired intestinal epithelium polarity(Melendez et al., 2013), clearly demonstrating a role for Cdc42 inepithelial cell proliferation and polarity.

Our observations in both the ureteric bud and metanephricmesenchyme are very similar to those demonstrated in pancreaticduct formation, where Cdc42 plays a cell-autonomous role inmicrolumen formation, in generation of apical polarity and in tightjunction formation (Kesavan et al., 2009). Therefore, we propose

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that the tubular lumen and cell differentiation lesions in the Hoxb7-cre:Cdc42flox/flox and Six2-cre:Cdc42flox/flox mice are primarilycaused by abnormalities in epithelial cell polarity due to defects inCdc42-dependent aPKC activation through Par6 (Goldstein andMacara, 2007) and abnormal apical actin cytoskeletal functioncaused by abnormal N-Wasp activation as described in othersystems (Rohatgi et al., 2000, 1999). The Cdc42 Six2-cre mice havea phenotype that is similar to mice lacking afadin: both are definedby their polarity defects and discontinuous lumen formation asevidenced by a decrease in Par3 staining in the pretubular aggregatesand/or renal vesicles, and the absence of a discrete lumen at the renalvesicle stage (Yang et al., 2013). The Six2-cre:Cdc42flox/flox

mutants do not pass through a true renal vesicle stage and smalldiscontinuous lumens develop in S-shaped bodies resulting in apersistent defect in luminal continuity. There are increased levels ofboth Par3 and aPKC in S-shaped bodies of Cdc42 Six2-cre mutantscompared with earlier stages. The mechanisms responsible for this‘recovery’ of the localization of Par complex components areunknown, but it is likely that partially redundant signaling pathwayscompensate for this process in the absence of Cdc42. Similar to theCdc42 mutants, afadin mutants also have defects in the actincytoskeleton as they are unable to recruit the actin subapical ring toform apical surfaces. In addition, afadin mutants fail to form Parcomplexes. Taken together, these results suggest that Cdc42 and

Fig. 8. Deleting Cdc42 from collecting ducts results incytoskeletal abnormalities. (A) Cdc42flox/flox and Cdc42−/−

collecting duct cells were allowed to adhere to collagen I for 1 h,after which they were treated with GDNF for 5 and 20 min. Thecells were then lysed and analyzed by western blotting for levels ofphosphorylated N-Wasp (pWasp), N-Wasp, phosphorylated Ret(pRet), phosphorylated ezrin (pEzrin), ezrin and β-actin.(B–E) Immunostaining of E15.5 Cdc42flox/flox (B,B′,B″,D,D′,D″)and Hoxb7-cre:Cdc42flox/flox (C,C′,C″,E,E′,E″) kidney sectionswith phalloidin (green in B–C″), the collecting duct marker DBA(red in B–C″), the nuclear marker DAPI (blue in B,C,D and E), thesub-apical marker ezrin (green in D–E″) and the collecting ductmarker cytokeratin (red in D–E″). B,C,D and E represent mergedimages; B′ and C′ are single images for phalloidin; B″ and C″ aresingle images for DBA; D′ and E′ are single images for ezrin; andD″ and E″ are single images for cytokeratin. Scale bars: 20 μm.

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afadin act in a similar pathway to promote polarization and lumenformation. Based on in vitro data from others (Kawakatsu et al.,2002; Kurita et al., 2013; Mandai et al., 2013; Nakanishi and Takai,2004), we speculate that afadin acts upstream of Cdc42 in thesignaling pathway; however, this still needs to be verified.We used Cdc42−/− collecting duct cells to demonstrate that Cdc42

is required for in vitro 3D tubulogenesis as well as collecting duct celladhesion, migration and proliferation on all extracellular matrices.Similar to our in vivo findings, these cells also have a majorabnormality in epithelial cell polarity with respect to formation ofadherens and tight junctions aswell as the Par3–Par6–aPKCcomplex.Our results are similar to those demonstrating a role for Cdc42 inpolarized mammary epithelial cell migration and branching, whereCdc42 was either overexpressed or knocked down in vitro (Duanet al., 2010), and overexpressed in the mouse mammary gland in vivo(Bray et al., 2013). In the kidney tubular epithelium, the functionalcharacteristics of Cdc42 have been explored in the well-characterizedpolarized MDCK cells. Utilizing overexpression and knockdowntechniques,Cdc42 has been shown to be required for lumen formationof tubules because it mediates exocytosis of large intracellularvacuoles that become the lumen and it also binds to the Par3–Par6–aPKC complex, which regulates tight junction formation andepithelial cell polarity (Martin-Belmonte et al., 2007, 2008; Martin-Belmonte and Mostov, 2007; Rodriguez-Fraticelli et al., 2010). InadditionCdc42 has been shown to play a role in localizing the exocystto the primary cilium of MDCK cells (Zhang et al., 2001; Zuo et al.,2011). The effects of deleting Cdc42 on MDCK cell tubulogenesisare much less severe than those seen in the Cdc42−/− collecting ductcells, which never progress to the cyst stage in 3D culture. These datasuggest very distinct roles for Cdc42 in tubulogenesis and lumenformation in collecting duct and MDCK cells, and that Cdc42 playsmultiple roles in tubule formation in different cell types.We demonstrate that the principal signaling defect observed in

Cdc42−/− collecting duct cells is their inability to activate N-Waspand ezrin in response to growth factors. There was also a majordefect of apical ezrin expression and localization in collecting ductsin vivo. Taken together, these data suggest that local recruitment ofactivated Cdc42 and its downstream effector, N-Wasp, can triggeractin polymerization in the apical domain of the epithelial cell(Castellano et al., 1999; Nakamura et al., 2000). This is consistentwith the well-defined role of Cdc42 in localizing the Par3–Par6–aPKC complex to the apical part of a polarized epithelial cell(Joberty et al., 2000; Welchman et al., 2007).In conclusion, we and others have shown that Cdc42 is required

for normal tubulogenesis in ureteric bud and metanephricmesenchyme development of the kidney (Choi et al., 2013;Reginensi et al., 2013). Although the morphological observationsare similar in all these studies, the mechanisms proposed differ. Wedemonstrate both in vivo and in vitro that the predominant role ofCdc42 in renal tubulogenesis, as in other branching organs like thelung (Wan et al., 2013) and the pancreas (Kesavan et al., 2009), is toregulate epithelial cell proliferation, polarity and the actincytoskeleton. Thus, in keeping with its pleiotropic effects, Cdc42regulates epithelial cell tubulogenesis by multiple mechanisms.This emphasizes the importance of studying its cell autonomousfunctions in different situations in epithelial cell biology.

MATERIALS AND METHODSMiceAll experiments were approved by the Vanderbilt University InstitutionalAnimal Use and Care Committee. Cdc42flox/flox mice, which were previouslydescribed (Wu et al., 2006), were crossed with the Hoxb7Cre mice (generous

gift of Andrew McMahon, Department of Stem Cell Biology andRegenerative Medicine, USC Keck School of Medicine, CA) (Yu et al.,2002) or Six2-cre mice (generous gift of Andrew McMahon) (Kobayashiet al., 2008). Micewere a F4–F6 generation towards the C57/B6 background.Aged-matched littermates Cdc42flox/flox mice were used as controls.

Morphologic and immunofluorescence analysisFor morphological and immunohistochemical analysis, kidneys wereremoved at different stages of development and fixed in 4% formaldehydeand embedded in paraffin. Paraffin tissue sections were stained with eitherhematoxylin and eosin or periodic acid Schiff’s (PAS) for morphologicalevaluation by light microscopy.

Immunofluorescence was performed as previously described (Yang et al.,2013). In brief, fixed sections (4% PFA for 2 h) were permeabilized with0.3% Triton X-100 in PBS (PBST) and blocked with 10% bovine serumalbumin (BSA) in PBST. Sections were incubated with primary antibodiesovernight (4°C), thenwith fluorophore-conjugated secondary antibodies andmounted with Vecatshield (Vector Laboratories). Antibodies used wereagainst Par3 (1:150, Millipore, 07-330), aPKCζ (1:400, Santa CruzBiotechnology, C-20) and phalloidin 647 and 488 (1:200, Invitrogen,42008A), YAP and phosphorylated YAP (1:500, Cell Signaling 4912, 4911,tyramide amplification), V-ATPase (1:500, Santa Cruz Biotechnology,20943), cytokeratin (1:500, Sigma-Aldrich, C2562), LTL (1:500, VectorLabs, B1325), NCAM (1:500, no antigen retrieval), E-cadherin (1:300,Invitrogen), DBA (1:500, Vector Labs, B1035), ezrin (1:500, Upstate 07-130), ZO1 (Santa Cruz Biotechnology, 33725), Aqp2 (Sigma-AldrichA7310), acetylated αtubulin (Cell Signaling, 5335P) and Six2 (Proteintech,11562-1). Secondary antibodies were purchased from JacksonImmunoResearch and Alexa-Fluor-conjugated antibodies from Invitrogen.

Cell proliferation in vivoKidney sections obtained at different embryonic stages were subjectedto immunohistochemical analysis using primary antibodies againstphosphorylated histone H3 (pHH3, 1:2000, Sigma-Aldrich, tyramideamplification), Six2 (1:500, Proteintech) and cytokeratin (1:500, Sigma-Aldrich). More than 250 Six2- or cytokeratin-positive cells were countedfrom each kidney at each stage and the percentage of pHH3 co-positive cellswere calculated. For each stage and genotype a minimum of four differentembryos were analyzed.

Generation of Cdc42−/− cellsCollecting duct cells were isolated from Cdc42fl/fl mice following themethodology described by Husted et al. (1988) and Cdc42 was deleted byinfecting the cells with an adenocre virus in vitro. Deletion of Cdc42 wasverified by immunoblotting for Cdc42. Clonal Cdc42-null cell lines weregenerated and similarity in their behavior was verified.

Tubulogenesis assayCollecting duct cells were grown in collagen or matrigel gels as previouslydescribed (Chen et al., 2004). Collecting duct cells (5×103) were seeded intothe gels, which were overlaid with 100 μl of medium and allowed to growfor 5 to 7 days. The gels were stained with Rhodamine–phalloidin (R415,1:2000, Molecular Probes) and the tubules were photographed using a ZeissAxio 510 confocal microscope (400×).

Cell spreadingCells were plated onto slides coated with collagen I (10 μg/ml) for 2 h afterwhich they were fixed, permeabilized, exposed to Rhodamine–phalloidin(1:5000) and visualized under a fluorescence microscope.

Cell adhesion and migration assaysCell adhesion and migration assays on different ECM components wereperformed as described previously (Chen et al., 2004). Four independentexperiments were performed in triplicate.

Cell proliferationProliferation assays were performed as previously described (Chen et al.,2004). Briefly, 5×103cells were plated on different ECM components in

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96-well plates and maintained in Dulbecco’s modified Eagle’s medium(DMEM) with 10% fetal bovine serum (FBS). After 12 h, the cells wereswitched to DMEM (2% FBS) for 24 h and then pulsed with 1 µCi/well[3H]thymidine (PerkinElmer Life Sciences). After another 24 h, the cellswere solubilized and radioactivity was measured using a scintillationcounter.

Cell polarityCells were grown on Transwell inserts consisting of polyvinylpyrolidone-free polycarbonate filters with 0.4 µm pores. After reaching confluency,cells were fixed in 4% formaldehyde and incubated with anti-ZO-1 (1:200;BD Transduction Laboratories) or anti-E-cadherin (1:1000; BDTransduction Laboratories) antibodies followed by the appropriate FITC-conjugated secondary antibody. Chamber slides were mounted and viewedusing a confocal microscope.

ImmunoblottingThe effect of glial-derived neurotropic factor (GDNF) on collecting ductcells was examined as previously described (Zhang et al., 2009). In brief,cells were trypsinized into serum-free DMEM and then plated on collagen I(10 μg/ml) for 45 min. GDNF (10 ng/ml) was added to the medium and thecells were lysed at different time points following growth factor stimulation.

For analysis on kidney tissues, the cortices or medullas were removed andlysed with T-Per buffer (ThermoScientific). Lysates were clarified bycentrifugation, and 30 μg total protein was electrophoresed onto a 10%SDS-PAGE gel and subsequently transferred to PVDF membranes.Membranes were blocked in 5% milk with TBS containing 0.1% Tween20 and then incubated with the different primary antibodies followed bythe appropriate horseradish peroxidase (HRP)-conjugated secondaryantibodies.

Immunoreactive bandswere identified using enhanced chemiluminescenceaccording to the manufacturer’s instructions. Antibodies against Cdc42(2466, 1:1000), ezrin (3142, 1:1000), phosphorylated ezrin (3144, 1:1000)and phosphorylated Ret (3221, 1:1000) were purchased from Cell Signaling;anti-PKCζ antibody was from Upstate (17264, 1:1000), anti-ParD3A (sc-98509, 1:1000) antibody was from Santa Cruz Biotechnology, antibodiesagainst N-Wasp (wp2001, 1:1000) and phosphorylated N-Wasp (wp2601,1:1000) were from ECMBiosciences, anti-ZO-1 (33-9100, 1:1000) antibodywas from Life Technologies and anti-E-cadherin antibody (610181, 1:2000)was from BD Biosciences

Cell fractionationTriton-insoluble (actin-rich) and Triton-soluble fractions of collecting ductcells were prepared as described previously (Elias et al., 2014). Briefly, cellswere incubated for 5 min with lysis buffer CS (50 mm Tris-HCl, pH 7.4,1.0% Triton X-100, 5 mm EGTA, and 10 μg/ml protease inhibitor mixture).Cell lysates were centrifuged at 15,600 g for 5 min at 4°C to sediment thehigh-density actin-rich fraction. The pellet was suspended in 200 μl of lysisbuffer D (0.3% SDS in 20 mm Tris-HCl buffer, pH 7.4, and 10 μg/mlprotease inhibitor mixture).

StatisticsThe Student’s t-test was used for comparisons between two groups, andanalysis of variance using Sigma Stat software was used for statisticaldifferences between multiple groups. P<0.05 was considered statisticallysignificant.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsBertha C. Elias, Amrita Das, Diptiben V. Parekh, Glenda Mernaugh, RebeccaAdams, Zhufeng Yang and Denise K. Marciano performed experiments for thepaper. Cord Brakebusch made the floxed Cdc42 mice. Ambra Pozzi, Bertha Elias,Denise K. Marciano, Thomas J. Carroll and Roy Zent wrote the paper.

FundingThis research was funded by VA Merit Reviews [grant numbers 1I01BX002025 toA.P., 1I01BX002196 to R.Z.]; by the National Institutes of Health [grant numbers

R01-DK083187 to R.Z., R01-DK075594 to R.Z., R01-DK383069221 to R.Z., R01-DK095761 to A.P., RO1-DK080004 to T.J.C., RO1-DK095057to T.J.C., R01-DK099478 to D.K.M., 5P30DK-079328 to T.J.C.]; and by March of Dimes grants toT.J.C. and D.K.M. Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.164509/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2015) 128, 4293-4305 doi:10.1242/jcs.164509

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